Gonorrhea is one of the most commonly reported bacterial infection in the United States. Over 3 milion cases are reported annually. See Morello et al., "Neisseria and Branhamella", Manual of Clinical Microbiology (5th Ed. 1991), pp. 258-276.
In order to treat successfully a disease caused by a bacterium, for example Neisseria gonorrhoeae, the rapid and accurate detection and identification of the disease-causing bacterium is required. The detection and identification has traditionally been accomplished by pure culture isolation and determination procedures that make use of knowledge of specimen source, growth requirements, visible (colony) growth features, microscopic morphology, staining reactions, and biochemical characteristics.
Conventional methods of detecting and identifying the causative agent of gonorrhea, N. gonorrhoeae, include the Gram-stain, culturing on selective agar medium, and cytochrome oxidase and carbohydrate utilization testing. Serological assays, including coagglutination and fluorescent antibody staining have also been described for the detection of N. gonorrhoeae. See, Morello et al., supra. Recently, the use of DNA probes for the diagnosis of N. gonorrhoeae has also been reported by Morello et al., supra. The Gram-stain and antibody-based tests are rapid (&lt;1 hour), but of low sensitivity (requiting at least 10.sup.4 CFU (colony forming units) bacteria per ml). Culture methods, while sensitive to approximately 2 CFU per ml, require overnight incubation.
The use of specific polynucleotide sequences as probes for the recognition of infectious agents is becoming a valuable alternative to problematic immunological identification assays. For example, PCT publication WO84/02721, published 19 Jul. 1984 describes the use of nucleic acid probes complementary to targeted nucleic acid sequences composed of ribosomal RNA, transfer RNA, or other RNA in hybridization procedures to detect the target nucleic acid sequence. Analogously, Miyada, C. G. and Born, T. L. (1991), Molecular and Cellular Probes 5: 327-35, used probes to detect N. gonorrhoeae. While the assay may provide greater sensitivity and specificity than known DNA hybridization assays, hybridization procedures which require the use of a complementary probe are generally dependent upon the cultivation and/or enrichment of a test organism and are, therefore, unsuitable for rapid diagnosis. Probes can be used directly on clinical specimens if a means of amplifying the DNA or RNA target is available.
There is a continued need for a simple, rapid, sensitive and specific diagnostic technique for the detection of N. gonorrhoeae in clinical samples.
In addition, patients infected with N. gonorrhoeae are often also infected with Chlamydia trachomatis. To minimize the number of diagnostic procedures to which a patient is subjected, as well as to minimize the cost of overall diagnosis, it would be highly desirable to have a simple, rapid and sensitive diagnostic technique for the simultaneous detection and identification of N. gonorrhoeae and C. trachomatis in one test procedure. The novel probes and techniques of the present invention which render it feasible to simultaneously detect N. gonorrhoeae and C. trachomatis is an additional feature of the present invention.
Use of the polymerase chain reaction ("PCR") has revolutionized the detection of a wide variety of bacterial, fungal, viral and parasitic pathogens. Enrichment and in vitro culturing of the pathogen is not required, and a relatively crude clinical specimen can provide the source of the nucleic acid for detection and diagnosis. PCR effects the targeted amplification of a specific nucleic acid sequence which dramatically increases the number of copies for detection and concomitantly reduces the complexity of the nucleic acid being analyzed.
The published sequence of a 1044 base pair fragment (ORF 1) of N. gonorrhoeae DNA is set forth in Miyada and Born, supra. Probes based on this ORF 1 sequence were shown to hybridize to 105 of 106 N. gonorrhoeae strains tested using a chromosomal dot blot format. Cross reactivity to other Neisseria species was only observed with N. mucosa, however this cross reactivity was eliminated with a high stringency wash. The protein coded for by ORF 1 was identified as having significant homology to the N. gonorrhoeae cytosine DNA methyltransferase gene (M. Ngo PII).